CN104164481A - Method for detecting transcription level of tree shrew xanthine dehydrogenase/oxidase gene through RT-PCR - Google Patents
Method for detecting transcription level of tree shrew xanthine dehydrogenase/oxidase gene through RT-PCR Download PDFInfo
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Abstract
The invention provides a method for detecting the transcription level of tree shrew xanthine dehydrogenase/oxidase gene through RT-PCR. The method comprises the following steps: carrying out PCR amplification by using a PCR primer combination with cDNA obtained after inverse transcription of total RNA of a sample as a template to obtain a ratio of the gray value of an amplification product of an XDH/XO gene fragment to the gray value of a PCR amplification product of an internal control gene GAPDH, calculating the relative expression value of XDH/XO mRNA under different physiological conditions according to the gray value ratio, and semi-quantitatively detecting the transcription level of tree shrew xanthine dehydrogenase/oxidase gene according to the calculated relative expression value in order to provide an effective way to study the functions of tree shrew xanthine oxidase and the influences of related drugs on the tree shrew xanthine oxidase. The method is suitable for the semi-quantitative RT-PCR detection, and has the advantages of low cost, simple operation, high repeatability low detection cost, and good specificity and sensitivity.
Description
Technical field
The present invention relates to a kind of detection method, especially a kind of method that detects tree shrew xanthine dehydrogenase/oxydase transcriptional level by semi-quantitative RT-PCR, belongs to field of molecular biotechnology.
Background technology
Xanthine dehydrogenase/oxydase (xanthine dehydrogenase/oxidase,
xDH/XO) be a kind of non-specific aerobic dehydrogenase, the last two steps of catalysis purine metabolism in purine metabolism process, from xanthine and xanthoglobulin, form uric acid metabolism product end product uric acid, in the metabolic process of uric acid, playing an important role, is a kind of very important enzyme in the interior uric acid metabolism of body.
Along with the raising of people's living standard, the sickness rate of hyperuricemia raises year by year in recent years, and normal and hypertension, hyperlipidaemia and diabetes etc. are followed appearance.Domestic and foreign literature report, hyperuricemia is obviously relevant to coronary heart disease, hypertension, diabetes, cerebral apoplexy etc., is its independent risk factor and the important prognosis prediction factor.The research of gout and hyperuricemia is just becoming the focus of medical medical circle.No matter be the screening of medicine, or the research of mechanism of causing a disease, all need research and the relevant medicine that falls blood uric acid of exploitation of selecting a kind of scientific and rational laboratory animal to carry out hyperuricemia pathogenesis.
Tree shrew (
tupaia belangeritree shrews) classification position is between Primates and Insectivora, and its evolution degree is high, and metabolism and gross anatomy more approach with people than animals such as dog, large mouse, anatomical structure is also similar to people, so it is as the widespread use in biological study of comparatively ideal laboratory animal.
In order to utilize the sibship laboratory animal approaching to the mankind---tree shrew is carried out research and the relevant medicine that falls blood uric acid of exploitation of hyperuricemia pathogenesis, need to set up a kind of relative quantification method that tree shrew xanthine dehydrogenase/oxydase mRNA expresses, with xanthine dehydrogenase in drugs effect tree shrew body/oxidasic transcriptional level, change, and then lay the first stone for studying XOD function and influence factor thereof.Sxemiquantitative RT-PCR is because its expense is low, common lab just can be accomplished, and the effective means of conduct research gene transcription level can be used to gene expression detection and changing condition thereof.Yet there are no both at home and abroad at present utilizes tree shrew to carry out the research of XOD/desaturase transcriptional level semi-quantitative method.
Summary of the invention
The object of this invention is to provide a kind of method that detects tree shrew xanthine dehydrogenase/oxidase gene transcriptional level by semi-quantitative RT-PCR, tree shrew xanthine dehydrogenase/oxidase gene is carried out to half-quantitative detection on transcriptional level.
For achieving the above object, the present invention completes by following technical proposal: the cDNA that the total RNA reverse transcription of the sample of take becomes is template, utilizes PCR combination of primers to carry out PCR amplification, obtains
xDH/XOthe gray-scale value of the amplified production of gene fragment and reference gene
gAPDHthe ratio of the gray-scale value of pcr amplification product, can calculate under different physiological situations according to the ratio of gained gray-scale value
xDH/XOrelative expression's value of mRNA, and according to big or small half-quantitative detection tree shrew xanthine dehydrogenase/oxidase gene transcriptional level of calculated relative expression's value.
Concrete scheme of the present invention is: a kind of semi-quantitative RT-PCR detects the method for tree shrew xanthine dehydrogenase/oxydase transcriptional level, it is characterized in that comprising the following steps:
1) design following primer:
Tree shrew
xDH/XOthe specificity upstream and downstream combination of primers of gene expression dose and corresponding nucleotides sequence are classified as:
XDH/XO F：5’-GTTGGAGGGAACATCATCACT-3’
XDH/XO R：5’-GCAGGGTCTTTCTGTAGCCA-3’；
Tree shrew as reference gene
gAPDHthe specificity upstream and downstream primer of gene and corresponding nucleotides sequence are classified as:
GAPDH F：5’-CAAGAAGGTAGTGAAGCAGGC-3，
GAPDH R 5’-TGTTGAAGTCGGAGGAGACC-3’
2) take total RNA of tree shrew fresh liver tissue extraction is template, routinely synthetic tree shrew liver organization cDNA the first chain that to obtain of reverse transcription;
3) take step 2) synthetic tree shrew liver organization cDNA the first chain of reverse transcription is template, with in step 1)
xDH/XOf and
xDH/XOr and
gAPDHf and
gAPDHr is Auele Specific Primer, respectively under following PCR amplification system: 25 μ L systems, Premix Taq 12.5 μ L, 10 μ mol/L forwards, each 1 μ L of reverse primer, cDNA template 2 μ L, 8.5 μ L deionized waters, carry out following pcr amplification: 94 ℃ of denaturation 5 min, 98 ℃ of sex change 10S, 58 ℃ of annealing 30S, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 10 min, obtain respectively
xDH/XOwith
gAPDHamplified production;
4) by step 3)
xDH/XOwith
gAPDHamplified production through 1.5% agarose gel electrophoresis, detect respectively routinely, obtain respectively the goal gene fragment consistent with designed goal gene size, and on gel image analyzers, obtain respectively gene histogram picture, with Quantity one software, obtained gene band is carried out to gray analysis, to obtain respectively
xDH/XOgene band gray-scale value and
gAPDHgene band gray-scale value;
5) according to step 4), obtain
xDH/XOgene band gray-scale value and
gAPDHthe ratio of gene band gray-scale value, calculates
xDH/XOmRNA relative expression value, can judge the changing conditions of tree shrew xanthine dehydrogenase/oxidase gene transcriptional level according to the size of this relative expression's value.
The consistent product of goal gene size in described step 4) and designed is:
xDH/XOthe PCR product size of gene is 150bp,
gAPDHthe PCR size of gene is 100bp, and agarose gel electrophoresis result as shown in Figure 2.
The PCR amplified production that described step 3) obtains passes through respectively following checking:
1) by the order-checking of business biotech firm, obtain respectively:
xDH/XOgene fragment amplification sequence is:
GACTCATCCT GTGTTCATGG CGAGTGTGGC CAAGCTGACC CTCGTGTCCA
GAGGCACCAG GAGAACTGTT CGAATGGATC ACACCTTCTT CCCTGGCTAC
AGAAAGACCC TGCA
Tree shrew reference gene
gAPDHfragment amplification sequence is:
AAAGAATTGA GCATCAGGCA TCCTGGGCTA CACTGAGCAC CAGGTGGTCT CCTCCGACTT CAACAA；
2) people who more above-mentioned order-checking gained nucleotide sequence is reported by DNAMAN software and NCBI respectively and macaque
xDH/XOgene
and GAPDHthe nucleotide sequence of gene carries out sequence analysis, and result shows: in the tree shrew liver organization that primer of the present invention increases
xDH/XOgene and the mankind's homology is 87.04%,
gAPDHwith the homology of macaque be 77.3%, illustrate that amplified object fragment is respectively tree shrew
xDH/XOgene fragment and
gAPDHgene.
Technological difficulties to be solved by this invention are:
Tree shrew is as a kind of Novel experimental animal, relevant both at home and abroad
xDH/XOgene order and
gAPDHgene order has no report, and the present invention is with the mankind's in NCBI gene pool
xDH/XOand macaque (
macaca mulatta)
gAPDHnucleotide sequence as a reference, with the multipair primer of Primer premier 5.0 software design, through RT-PCR amplification, is groped the optimum annealing temperature of pcr amplification according to the feature of primer, filter out detection tree shrew described above
xDH/XOthe specificity upstream and downstream combination of primers of genetic expression and pcr amplification condition, obtained special
xDH/XOgene fragment and reference gene
gAPDHfragment, and by obtained special
xDH/XOgene fragment and reference gene
gAPDHthe order-checking of fragment and sequential analysis, further confirmed the specificity of the PCR combination of primers that provides to can be applicable to RT-PCR method half-quantitative detection tree shrew xanthine dehydrogenase/oxidase gene transcriptional level.
The present invention has following advantages and effect:
1) adopt such scheme, can just can easily carry out tree shrew xanthine dehydrogenase/oxidase gene in common lab and transcribe, and on transcriptional level, tree shrew xanthine dehydrogenase/oxidase gene be carried out to half-quantitative detection.
2) primer disclosed in this invention, by well known to a person skilled in the art that chemical synthesis process carries out after DNA synthesizes obtaining, can be realized tree shrew with this primer sequence
xDH/XOgene and reference gene
gAPDHspecific amplification, non-goal gene in material is not had to amplified signal, by primer of the present invention, can implement half-quantitative detection to the changing condition of tree shrew xanthine dehydrogenase/oxidase gene transcriptional level, method is simple, repeatable high, testing cost is low.And primer specificity of the present invention is strong, can not be subject to the interference of other genes, for studying function and the influence factor of tree shrew xanthine dehydrogenase/oxidase gene, provide effective instrument.
Accompanying drawing explanation
Fig. 1 is the agarose electrophoresis figure of RNA integrity mensuration';
Fig. 2 is
xDH/XOwith
gAPDHgene amplification agarose electrophoresis figure;
Fig. 3 is ALLO in the hyperuricemia tree shrew hepatic tissue due to OA
xDH/XOthe agarose electrophoresis figure of the impact of mrna expression level;
Fig. 4 is ALLO in the hyperuricemia tree shrew hepatic tissue due to OA
xDH/XOthe impact of mrna expression level.
Embodiment
The experimental technique using in following embodiment, if no special instructions, is ordinary method.
The material reagent using in following embodiment etc., if no special instructions, are commercial sources and obtain.
The laboratory animal of 1, using in the present embodiment:
Middle remote tree shrew the western regions of the Yunnan Province subspecies, common grade is raised, 20 ℃～25 ℃ of feeding environment temperature, humidity 40%～70%, normally feeds, and freely drinks water.Body weight is 110-150g, 1～3 years old age, and male and female half and half, conventional unified loin chop feed is fed.
2, various solution preparations
The preparation of 1% Xylo-Mucine (being abbreviated as 1%CMC-Na): take 3g CMC-Na(Kunming Cheng Yue scientific & technical corporation and produce), put into the Erlenmeyer flask of 500ml, slowly add the sterile purified water that 100ml boils to expand, slowly add again 100ml sterile purified water and on electric furnace, boil dissolving, finally add the sterile purified water that boils and dissolve constant volume to 300ml, normal temperature is preserved, now with the current.
Different concns Oteracil Potassium (oxonic acid potassium salt), be abbreviated as the allopurinol (allopurinol) of OA solution, different concns, be abbreviated as the preparation of ALLO solution, press 10ml/kg volume, to reach each every animal abdominal injection volume, be controlled at 1-2ml left and right, be convenient to animal and absorb preferably.
According to 10mg/ml, 5mg/ml different concns, carry out respectively OA solution preparation, OA selects Sigma-Aldrich company product, Lot# STBC64180, purity >=97%.Compound method: electronic balance accurately takes OA 150 mg, 15 mg in 15ml centrifuge tube, adds 15ml 1%CMC-Na solution, stirs with glass stick, is made into the OA suspension solution of 10mg/ml, 5mg/ml, now with the current.During use, press respectively the kg body weight of thing and calculate desired concn volume.During use, press respectively the kg body weight of thing and calculate selection respective concentration suspension calculating desired concn volume, if tree shrew weight is 100g, injected dose is 100mg/kg, selects respective concentration 10mg/mL suspension 1mL abdominal injection.
According to 3mg/mL, 2mg/mL and 0.4mg/mL different concns, carry out respectively the preparation of ALLO, ALLO selects the biological company limited of Lay products of Nanjing, purity >=98%, compound method: electronic balance accurately takes ALLO 45mg, 30mg and 6mg respectively in independent 15mL centrifuge tube, all add 15mL 1%CMC-Na solution, with glass stick, stir, be made into the ALLO suspension solution of 3mg/mL, 2mg/mL and 0.4mg/mL, now with the current.During use, press respectively the kg body weight of thing and calculate selection respective concentration suspension calculating desired concn volume, if tree shrew weight is 100g, injected dose is 30mg/kg, selects respective concentration 3mg/mL suspension 1mL abdominal injection.
3, instrument and consumptive material
Sigma high speed freezing centrifuge, the precise electronic balance ME235S of Sartoyius company; U.S. bio-rad company gel imaging analysis system; The U.S. PCR of bio-rad company instrument:; The PowerPac Basic of U.S. bio-rad company electrophoresis apparatus; The Japan Tomy SS-325 of company autoclaving case; The U.S. ND-1000 of DanoDrop company ultraviolet spectrophotometer.The Gilson liquid-transfering gun of 1000ul, 200ul, 20ul, 10ul, 2ul; 2ml EP pipe, 500ul EP pipe; 1000ul suction nozzle, 200ul suction nozzle, 10ul suction nozzle.
4, the grouping of laboratory animal
Choose 30 of normal tree shrews, 16 of ♀, 14 of ♂, body weight 120.6 ± 10.6g, is divided into following 5 groups at random, 6 every group, is respectively: OA group, 1 group of OA+ALLO, 2 groups of OA+ALLO, ALLO group, control group.Abdominal injection, injecting method is:
0 hour to OA group, 1 group of OA+ALLO and 2 groups of injection OA 80mg/kg of OAOA+ALLO, to ALLO group injection ALLO 4 mg/kg, the 1%CMC-Na of control group injection equal volume;
After 0.5 hour, OA+ALLO 1 treated animal is injected ALLO 4 mg/kg, OA+ALLO 2 treated animals are injected to ALLO 30mg/kg.
5, experimental technique
The live body operation sampling of 5.1 tree shrew fresh liver tissues
The fresh liver that after administration, 2h gets respectively the every treated animal of above-mentioned group is organized each 0.1g, puts into rapidly liquid nitrogen and preserves, for next step experiment.
The extraction of the total RNA of 5.2 liver organization
The Trizol test kit specification sheets of producing by U.S. invitrogen company extracts.The fresh liver that each group is obtained is organized the mortar of putting into respectively precooling, and limit adds liquid nitrogen limit grinds, until make hepatic tissue become white powder, the abundant cracking of Trizol reagent that adds respectively 1ml, fully grinds after 10min, until become clarified liq, respectively liquid rotating is moved in the EP pipe of corresponding 1.5ml, static 5min, 4 ℃, the centrifugal 5min of 13500r/m, get supernatant liquor and add equal-volume chloroform, standing 10min after upper and lower jolting 20 times,, shifts water after the centrifugal 30min of 13500r/m by 4 ℃; Add respectively the upper and lower jolting of isopyknic pre-cold isopropanol 20 times, then standing 20min, more centrifugal 20min, supernatant liquid inclines, now to have a small amount of white precipitate be RNA at visible each pipe end, adds after 1ml 75% ethanol elution, at 4 ℃ of centrifugal 5min of 13500r/m, supernatant liquid inclines, in drying at room temperature, after most of ethanol volatilizes, add 30ul DEPC water dissolution, obtain respectively total RNA sample that each extracts, and carry out mark and put into-80 ℃ of Refrigerator stores standby.
5.3 RNA concentration determination and purity check
Total RNA sample that 5.2 steps are extracted is measured through Nanodrop-1000 ultramicron nucleic acid determination instrument respectively, required addition when determining reverse transcription, each A260/ A280nm value of organizing sample all between 1. 8～2. between 0, illustrate that purity is higher.
5.4 RNA integrity detection
Get at random total RNA sample 2ul that two pipe 5.2 steps extract and carry out 1.5% agarose gel electrophoresis (120V 400mA) 20min, under gel imaging analysis system, observe 28S, 18S and the brightness of 5S RNA band, analyze the integrity of RNA.Result can be observed 28 S, 18 S, 5 S tri-bands, sees Fig. 1, illustrates that extracted total RNA is good without degraded and integrity, can be further used for subsequent experimental.
The reverse transcription of 5.5 RNA
Press Roche company's T ranscriptor First Strand cDNA specification sheets (04897030001) reverse transcription; Concrete steps are as follows:
Get respectively total RNA sample that 5.2 steps are extracted, with DEPC water, be diluted to 1000ng/ul respectively, by Roche the first chain cDNA synthetic agent box Transcriptor First Strand cDNA Synthesis Kit specification sheets operation, as shown in table 1; Making respectively each population of samples amass is 13 μ l, and under 65 ° of C conditions, reacting by heating pipe 10min makes the sex change of template-primer mixture, immediately reaction tubes is placed in to cooled on ice afterwards.Remaining RT reaction solution component in adding table 2 respectively in the test tube that contains template-primer mixture at each; The final volume of each sample is 20 μ l, in each reaction tubes, reagent is carefully mixed, and finally in PCR instrument, reacts, and its program is as follows: 25 ° of C 10min, 55 ° of C 30min, 85 ° of C 5min, 4 ℃ of ∞.Each the reverse transcription product cDNA obtaining is placed in to-30 ° of C and preserves, standby.
Table 1 RNA reverse transcription the 1st step
Table 2 RNA reverse transcription the 2nd step
5.6 design of primers and synthetic
Because tree shrew is as a kind of Novel experimental animal, both at home and abroad about tree shrew
xDH/XOgene order has no report, and the present invention is with the mankind's in NCBI gene pool
xDH/XOand macaque (macaca mulatta)
gAPDHas a reference, with Primer premier 5.0 software design primers, through Beijing, white Imtech is synthetic for nucleotide sequence,
gAPDHas reference gene.
The tree shrew of 5.7 the present embodiment
xDH/XOgene expression dose quantitatively uses primer sequence and clip size in Table 3.
Table 3 primer sequence and clip size
Determining of 5.8 pcr amplification systems and annealing temperature
By the biological product P CR of the company limited reagent of TaKaRa (Premix Taq Code No. RR003A), carry out respectively
xDH/XOgene and
gAPDHthe pcr amplification of gene, PCR amplification system is as follows: 25 μ L systems, Premix Taq 12.5 μ L, 10 μ mol/L forwards, each 1 μ L of reverse primer, cDNA template 2 μ L, 8.5 μ L deionized waters, carry out following pcr amplification: 94 ℃ of denaturation 5 min, 98 ℃ of sex change 10S, 58 ℃ of annealing 30S, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 10 min, obtain respectively
xDH/XOwith
gAPDHamplified production, amplified production is shown in Fig. 2 through agarose electrophoresis detected result.
5.9 PCR product direct Sequencing checkings
xDH/XOamplified production and
gAPDHgene amplification product send the order-checking of the white Imtech in Beijing, and sequencing result is as follows:
Tree shrew
xDH/XOgene fragment amplification sequence is:
GACTCATCCT GTGTTCATGG CGAGTGTGGC CAAGCTGACC CTCGTGTCCA
GAGGCACCAG GAGAACTGTT CGAATGGATC ACACCTTCTT CCCTGGCTAC
AGAAAGACCC TGCA
Tree shrew reference gene
gAPDHfragment amplification sequence is:
AAAGAATTGA GCATCAGGCA TCCTGGGCTA CACTGAGCAC CAGGTGGTCT CCTCCGACTT CAACAA
5.10 gene sequencing interpretation of result and checkings
Sequencing result compares to goal gene and reference gene nucleotide sequence and the mankind (GI1314286) of NCBI report and the homology of macaque (NM_001195426) cDNA nucleotide sequence by DNAMAN software, and result shows and draws in tree shrew liver organization
xDH/XOgene and the mankind's homology is 87.04%,
gAPDHwith the homology of macaque be 77.3%.Illustrate that amplified object fragment is respectively tree shrew
xDH/XOgene fragment and
gAPDHgene.
5.11 ALLO are in the hyperuricemia tree shrew hepatic tissue due to oxygen potassium cyanate
xDH/XOthe half-quantitative detection of the variation of mrna expression level
The RNA that the fresh liver tissue that each group obtains is above mentioned, after RT-PCR amplification, amplified production carries out imaging after 1.5% agarose gel electrophoresis on gel image analyzers, and with Quantity one software, band is carried out to gray analysis, calculates relative expression quantity.By
xDH/XOthe gray scale of gene product with
gAPDHthe ratio of the gray scale of PCR product, calculates different groups
xDH/XOrelative expression's value of mRNA.
Experimental data in the present embodiment adopts mean ± standard error (Mean ± SD) to represent, relatively selects between two the methods such as t-test between group, and application SPSS13.0 software carries out statistical study.During P<0.01, being utmost point significant difference, is significant difference during P<0.05, and P>0.05 is without significant difference.
Fig. 3 has shown that ALLO is in the hyperuricemia tree shrew hepatic tissue due to oxygen potassium cyanate
xDH/XOthe agarose electrophoresis figure of the expression of mRNA;
As shown in Fig. 4 and table 4, in OA group
xDH/XOgene relative expression value the highest, be 6.19, relatively there is significant difference (P ﹤ 0.01) with control group, all the other organize no difference of science of statistics (P ﹥ 0.05).Abdominal injection Oteracil Potassium 2h can significantly raise in tree shrew liver organization
xDH/XOmrna expression level, after injection Oteracil Potassium 0.5h, then in the tree shrew liver organization after abdominal injection allopurinol 1.5h
xDH/XOmrna expression level changes and control group indifference, injects separately after allopurinol 2h in tree shrew liver organization
xDH/XOmrna expression level changes and control group indifference.Prompting tree shrew is under high lithemia state
xDH/XOmrna expression level significantly increases.Allopurinol can cause under high lithemia state
xDH/XOmRNA lowers, and can not lower normal tree shrew
xDH/XOmrna expression level.
Table 4: in different dosing group tree shrew hepatic tissue
xDH/XOthe expression of mRNA
Note:
± S, n=2. and control group be * P < 0.01 relatively.Compare with OA group
#p < 0.01.
6, sxemiquantitative RT-PCR is the effective means of studying at present gene transcription level, because its expense is low, common lab easily accomplishes often to be used, can be used to gene expression detection and changing condition thereof, but in order to improve semiquantitative accuracy and reliability, should note problem in actual experiment: when reverse transcription and PCR, want the amount of Quality control RNA and template cDNA to improve the confidence level of measured value.
7, RT-PCR method except require goal gene and internal reference because of reverse transcription and PCR condition in full accord, compare and preferably with criticizing, carry out RT-PCR between sample, for detecting pcr amplification product with gel image scanning, the electrophoretic separation of carrying out on same gel preferably, makes to compare background consistent;
8, sxemiquantitative RT-PCR detection system must prevent from being mixed in the false positive that the genomic dna in RNA brings.For getting rid of possible pollution, in experiment, two negative controls can be set simultaneously, the one, the mRNA of take directly carries out PCR reaction as template, and another group does not add any template, if contain genomic dna, has band and occurs.
sequence table
XDH/XO gene specific upstream primer:
XDH/XO F：5’-GTTGGAGGGAACATCATCACT-3’
XDH/XO gene specific downstream primer:
XDH/XO R：5’-GCAGGGTCTTTCTGTAGCCA-3’
Reference gene GAPDH specificity upstream primer:
GAPDH F：5’-CAAGAAGGTAGTGAAGCAGGC-3
Reference gene GAPDH specificity upstream and downstream primer:
GAPDH R：5’-TGTTGAAGTCGGAGGAGACC-3’
The nucleotide sequence of the tree shrew XDH/XO gene fragment increasing:
GACTCATCCT GTGTTCATGG CGAGTGTGGC CAAGCTGACC CTCGTGTCCA
GAGGCACCAG GAGAACTGTT CGAATGGATC ACACCTTCTT CCCTGGCTAC
AGAAAGACCC TGCA
The nucleotide sequence of the tree shrew reference gene GAPDH fragment increasing:
AAAGAATTGA GCATCAGGCA TCCTGGGCTA CACTGAGCAC CAGGTGGTCT CCTCCGACTT CAACAA；
sequence table
XDH/XO gene specific upstream primer:
XDH/XO F：5’-GTTGGAGGGAACATCATCACT-3’
XDH/XO gene specific downstream primer:
XDH/XO R：5’-GCAGGGTCTTTCTGTAGCCA-3’
Reference gene GAPDH specificity upstream primer:
GAPDH F：5’-CAAGAAGGTAGTGAAGCAGGC-3
Reference gene GAPDH specificity upstream and downstream primer:
GAPDH R：5’-TGTTGAAGTCGGAGGAGACC-3’
The nucleotide sequence of the tree shrew XDH/XO gene fragment increasing:
GACTCATCCT GTGTTCATGG CGAGTGTGGC CAAGCTGACC CTCGTGTCCA
GAGGCACCAG GAGAACTGTT CGAATGGATC ACACCTTCTT CCCTGGCTAC
AGAAAGACCC TGCA
The nucleotide sequence of the tree shrew reference gene GAPDH fragment increasing:
AAAGAATTGA GCATCAGGCA TCCTGGGCTA CACTGAGCAC CAGGTGGTCT CCTCCGACTT CAACAA；
Claims (1)
1. RT-PCR detects a method for tree shrew xanthine dehydrogenase/oxidase gene transcriptional level, it is characterized in that comprising the following steps:
1) design following primer:
Tree shrew
xDH/XOthe specificity upstream and downstream combination of primers of gene expression dose and corresponding nucleotides sequence are classified as:
XDH/XO F：5’-GTTGGAGGGAACATCATCACT-3’
XDH/XO R：5’-GCAGGGTCTTTCTGTAGCCA-3’；
Tree shrew as reference gene
gAPDHthe specificity upstream and downstream primer of gene and corresponding nucleotides sequence are classified as:
GAPDH F：5’-CAAGAAGGTAGTGAAGCAGGC-3，
GAPDH R：5’-TGTTGAAGTCGGAGGAGACC-3’；
2) take total RNA of tree shrew fresh liver tissue extraction is template, routinely synthetic tree shrew liver organization cDNA the first chain that to obtain of reverse transcription;
3) take step 2) synthetic tree shrew liver organization cDNA the first chain of reverse transcription is template, with in step 1)
xDH/XOf and
xDH/XOr and
gAPDHf and
gAPDHr is Auele Specific Primer, respectively under following PCR amplification system: 25 μ L systems, Premix Taq 12.5 μ L, 10 μ mol/L forwards, each 1 μ L of reverse primer, cDNA template 2 μ L, 8.5 μ L deionized waters, carry out following pcr amplification: 94 ℃ of denaturation 5 min, 98 ℃ of sex change 10S, 58 ℃ of annealing 30S, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 10 min, obtain respectively special
xDH/XOwith
gAPDHamplified production;
4) step 3) is obtained
xDH/XOwith
gAPDHamplified production through 1.5% agarose gel electrophoresis, detect respectively routinely, obtain respectively goal gene fragment, and on gel image analyzers, obtain respectively gene histogram picture, and with Quantity one software, obtained gene band is carried out to gray analysis, obtain respectively
xDH/XOgene band gray-scale value and
gAPDHgene band gray-scale value;
5) according to step 4), obtain
xDH/XOgene band gray-scale value and
gAPDHthe ratio of gene band gray-scale value, calculates
xDH/XOmRNA relative expression value, judges tree shrew xanthine dehydrogenase/oxidase gene transcriptional level according to the size of this relative expression's value.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106498042A (en) * | 2016-10-15 | 2017-03-15 | 中国医学科学院医学生物学研究所 | The method that RT qPCR detect Wistar rat xanthine dehydrogenases/lysyloxidase gene transcriptional level |
| CN109750052A (en) * | 2018-12-12 | 2019-05-14 | 中国医学科学院医学生物学研究所 | Tree shrew xanthine oxidase fluidized dehydrogenation enzyme XDH/XO cDNA full length sequence and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106498042A (en) * | 2016-10-15 | 2017-03-15 | 中国医学科学院医学生物学研究所 | The method that RT qPCR detect Wistar rat xanthine dehydrogenases/lysyloxidase gene transcriptional level |
| CN109750052A (en) * | 2018-12-12 | 2019-05-14 | 中国医学科学院医学生物学研究所 | Tree shrew xanthine oxidase fluidized dehydrogenation enzyme XDH/XO cDNA full length sequence and application |
| CN109750052B (en) * | 2018-12-12 | 2024-03-19 | 中国医学科学院医学生物学研究所 | Tree shrew xanthine oxidative dehydrogenase XDH/XO cDNA full-length sequence and application thereof |
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